Protein Analysis Using A Polymethine Marker Dye

ABSTRACT

A kit for optically detecting proteins, in particular lipoproteins, in a sample, the kit comprising: a chip for performing a separation of the proteins, in particular of the lipoproteins, wherein the chip comprises at least one well for receiving the sample, and a separation channel coupled to the at least one well and being adapted for separating different compounds and a marker dye which contains a polymethine of the general formula (I) wherein Z is a substituted derivative of benzooxazole, benzodiazole, 2,3,3-trimethylindolinine, 2,3,3-trimethyl-4,5-benzo-3H-indolinine, 3- and 4-picoline, lipidine, chinadine and 9-methylacridine derivatives with the general formula IIa or IIb or IIc and wherein X is selected from the group consisting of O, S, Si, N-alkyl and C(alkyl) 2 , n is 0, 1 , 2 or 3, R 1  to R 14  are independently selected from the group consisting of hydrogen, alkyl, alkoxy, cycloalkyl, linear or branched alkenyl, cycloalkenyl, aryl, heteroaryl, heterocycle, hydroxy, carboxy, amine, alkyl-substituted amine and cyclic amine and/or two or more fragments in ortho-position to each other, for example R 10  and R 11  or R 4 , R 5  and R 6 , together form another cycloalkyl ring or ring system, heterocyclic ring or ring system, heteroaryl ring system or aromatic ring or ring system.

BACKGROUND ART

The present invention relates to a kit for optically detecting proteins,to a method for optically detecting proteins, to a method of analysingproteins and to the use of polymethines for optically detecting proteinsin a sample.

Determination of the circulating levels of plasma lipoproteins isimportant in the diagnosis of primary and secondary disorders of lipidtransport and in risk assessment for arteriosclerosis and coronaryartery disease. In the fasting state, three main lipoprotein classeshave been identified: VLDL (very low density lipoproteins), LDL (lowdensity lipoproteins) and HDL (high density lipoproteins), each of whichdiffers in size and density, and in lipid and apolipoproteincomposition.

It is well established that there is a positive correlation between riskof premature coronary heart disease and total plasma cholesterol andplasma LDL-cholesterol (LDL-C). There is also a correlation betweendecreased HDL-cholesterol (HDL-C) and increased plasma TG(triglycerides). Heterogeneity in the size and density of LDL is welldocumented and has also been shown to have clinical relevance. Smalldense LDL (Pattern B) has an increased relative risk compared with largelight LDL (Pattern A). One of the most prevalent lipid/lipoproteinpatterns associated with risk of coronary artery disease is theatherogenic lipoprotein phenotype (ALP), which is characterized bymoderately raised plasma TG, low levels of HDL-C, elevated total andLDL-C, and small, dense LDL particles. Although methods are available inthe clinical laboratory for measurement of HDL, LDL and VLDL, methodsfor the identification of the predominant LDL subclass are technicallydifficult and time-consuming.

The main methods for separation and analysis of the plasma lipoproteinlevels, based on differences in physical properties, includeultracentrifugation, electrophoresis and differential precipitation. Ofthese, ultracentrifugation is seen in the prior art as the “goldstandard” for analysis of plasma lipoproteins and potentially providesthe greatest amount of information, as the lipid and apolipoproteincompositions of the separated lipoproteins can be analysed.

Methods for density gradient centrifugation have generally been based onsalt solutions, and include sequential flotation with adjustment of thedensity of the plasma and infranatants after each centrifugation step,or centrifugation on discontinuous or continuous gradients. The use ofsalt gradients has a number of disadvantages. These are technicallydifficult to prepare and relatively unstable, and reproducibility isdifficult to achieve. In addition, prolonged centrifugation is oftennecessary to float the lipoproteins into the gradients and the high saltconcentrations can modify the protein structure and lead to loss ofapolipoproteins from the lipoprotein fractions. For further analysis ofthe lipoprotein fractions, it is usually necessary to remove the salt,e.g., by dialysis. This results in loss of material and poor recoveries.

Hence, it would be desirable to provide improved methods for analysinglipoproteins and in particular for characterizing sub-class patterns ofthe lipoproteins in order to better characterize the atherogenic risk ofa patient and to obtain more information for managing patients.

DISCLOSURE

It is an object of the invention to provide improved kits and methodsfor detecting and analysing proteins, in particular lipoproteins. Theobject is solved by the independent claims. Preferred embodiments areshown by the dependent claims.

According to one embodiment of the present invention, a kit foroptically detecting proteins, in particular lipoproteins, in a sample isprovided. The kit comprises a chip for performing a separation of theproteins, in particular of the lipoproteins, wherein the chip comprisesat least one well for receiving the sample and a separation channelcoupled to the at least one well and being adapted for separatingdifferent compounds. According to this embodiment, the kit furthercomprises a marker dye which contains a non-symmetrical polymethinecomprising a substituted ω-(benz[b]pyran-4-ylidene)alk-1-enyl) unit ofthe general formula I

wherein Z is a substituted derivative of benzooxazole, benzodiazole,2,3,3-trimethylindolinine, 2,3,3-trimethyl-4,5-benzo-3H-indolinine, 3-and 4-picoline, lipidine, chinadine and 9-methylacridine derivativeswith the general formula IIa or IIb or IIc

and wherein

X is selected from the group consisting of O, S, Si, N-alkyl andC(alkyl)₂,

n is 0, 1, 2 or 3,

R¹ to R¹⁴ are independently selected from the group consisting ofhydrogen, alkyl, alkoxy, cycloalkyl, linear or branched alkenyl,cycloalkenyl, aryl, heteroaryl, heterocycle, hydroxy, carboxyl, amine,alkyl-substituted amine and cyclic amine and/or two or more fragments inortho-position to each other, for example R¹⁰ and R¹¹ or R⁴, R⁵ and R⁶,together form another cycloalkyl ring or ring system, heterocyclic ringor ring system, heteroaryl ring system or aromatic ring or ring system.

In particular, the kit is advantageous for the analysis of lipoproteins,in that the marker dye as defined above has a high affinity for thehydrophobic inner compartment of lipoprotein particles, with little orno staining activity for other types of proteins or nucleic acids. Bymixing, e.g. human serum samples with this dye and subsequentelectrophoretic analysis in a microfluidic chip, the high densitylipoprotein (HDL) sub-fraction of lipoproteins can be baseline separatedfrom the low density lipoprotein (LDL) sub-fraction. Moreover, the HDLsub-fraction can be resolved into at least four to five individualsub-populations that can be quantitatively analysed. This kit enablesrapid and reproducible analysis for, e.g. patient serum samples forlipoprotein class distribution, i.e. percentage of HDL versus LDL, andalso for HDL and LDL sub-patterns.

According to a further embodiment of the present invention, a method ofanalysing proteins, in particular lipoproteins, in a sample is provided.The method comprises a step in which the proteins, in particular thelipoproteins, are separated in at least one dimension. The methodfurther comprises a step in which the proteins, in particular thelipoproteins, are labelled with a marker dye containing a polymethine ofthe general formula I as defined above. The method further comprises astep of optically detecting the separated and labelled proteins, inparticular the labelled lipoproteins.

Embodiments of the present invention further relate to a method foroptically detecting lipoproteins in a sample, wherein the methodcomprises a step of labelling lipoproteins with a marker dye containinga polymethine of the general formula (I) as defined above.

Finally, embodiments of the present invention relate to the use of apolymethine of the general formula I as defined above for opticallydetecting lipoproteins and/or for the analysis of lipoprotein classdistribution and/or for the analysis of HDL and/or LDL subclass patternsin a sample by labelling the proteins with the polymethine of thegeneral formula I.

Embodiments of the invention can be partly or entirely embodied orsupported by one or more suitable software programs, which can be storedon or otherwise provided by any kind of data carrier and which might beexecuted in or by any suitable data processing unit. Software programsor routines can be preferably applied to the method of analysingproteins, e.g. in the step of detecting the labelled lipoproteins or ina step of calibrating the obtained signals or converting them into agel-like image. For example, calibration steps according to anembodiment of the invention can be realized by a computer program, i.e.by software, or by using one or more special electronic optimisationcircuits, i.e. in hardware, or in hybrid form, i.e. by means of softwarecomponents and hardware components.

Further exemplary embodiments of the kit will be described below.However, these embodiments also apply for the method of analysinglipoproteins, for the method of optically detecting lipoproteins and forthe use of polymethines of the general formula I for optically detectinglipoproteins and/or for the analysis of lipoprotein class distributionand/or for the analysis of HDL and/or LDL subclass patterns.

According to a preferred embodiment of the kit, the separation channelis adapted for separating different compounds electrophoretically,chromatographically or electrochromatographically.

According to a further preferred embodiment of the kit, the separationchannel is adapted for separating different compoundselectrophoretically by electrophoresis selected from the groupconsisting of SDS polyacrylamide electrophoresis (SDS-PAGE), capillaryelectrophoresis and micro-channel/microfluidic channel electrophoresis.

According to a further preferred embodiment of the kit, the chip furthercomprises an element for applying an electrical field across theseparation channel.

According to a further preferred embodiment of the kit, the chipcomprises a material selected from the group consisting of glass,quartz, silica, silicon, and polymers.

According to a further preferred embodiment, the kit further comprises aseparation gel.

According to a further preferred embodiment of the kit, the separationgel is selected from the group consisting of polyacrylamide,polydimethylacrylamide, polyethylene oxide, and polyvinyl pyrrolidoneand is preferably polydimethylacrylamide.

According to a further preferred embodiment, the kit further comprises acalibration sample.

According to a further preferred embodiment of the kit, the calibrationsample is a “ladder”.

At least one of the substituents R¹ to R¹⁴ can also be a solubilising orionisable or ionised substituent like cyclodextrine, sugar, SO₃ ⁻, PO₃²⁻, COO⁻, or NR₃ ⁺ which determines the hydrophilic properties of thesedyes. Such a substituent may be bound to the marker dye by means of aspacer group. For example, said solubilizing or ionisable group is boundvia an aliphatic or heteroaliphatic group.

Further, it is preferred that at least one of the substituents R¹ to R¹⁴is a reactive group which is capable of reacting with a protein orlipoprotein to form a covalent or non-covalent bond. Such a substituentcan also be bound to the dye by means of a spacer group. Examples forsuch reactive groups are selected from the group consisting of anN-hydroxysuccinimidester group, a maleimide group and a phosphoamiditegroup.

According to a further preferred embodiment, R¹ to R¹⁴ are independentlyselected form the group consisting of hydrogen, chlorine, bromine, andan aliphatic or mononuclear aromatic group, each having at most 12carbon atoms which may contain as a substituted group in addition tocarbon and hydrogen up to 4 oxygen atoms and 0, 1 or 2 nitrogen atoms ora sulfur atom or a sulfur and a nitrogen atom or represent an aminofunction, having a nitrogen atom to which there is bound hydrogen or atleast one substituent having up to 8 carbon atoms, said substituentbeing selected from the group consisting of carbon, hydrogen and up totwo sulfonic acid groups.

According to a further preferred embodiment, any of the groups R¹ to R¹⁴is aliphatic and contains from 1 to 6 carbon atoms.

Further, it is preferred that R¹ is a substituent which has a quaternaryC-atom in α-position relative to the pyran ring. Examples for suchsubstituents are t-butyl (—C(CH₃)₃), phenyl and adamantyl(—C₁₀H₁₅/tricyclo[3.3.1.1^(3,7)]decyl). It is particularly preferredthat R¹=—(CH₃)₃.

According to a further preferred embodiment, R²R³, R⁴, R⁶, R⁷, R⁸, R⁹,R¹⁰, R¹¹, R¹² and/or R¹³ is hydrogen.

According to a further preferred embodiment, R⁵ is an amine oralkyl-substituted amine. It is particularly preferred thatR⁵=—N(CH₂CH₃)₂.

According to an alternative embodiment, R⁴ and R⁵ form a saturated,partially saturated or unsaturated, substituted or un-substitutedheterocyclic ring, preferably a six-membered heterocyclic ringcontaining one or more heteroatoms, preferably one or more nitrogenatoms, more preferably one nitrogen atom. Most preferably, the nitrogenatom of the heterocyclic ring corresponds to R⁵ and/or is substituted,e.g., by an ethyl group. It is further preferred that the heterocyclicring contains one double bond.

According to a further alternative embodiment, R⁴, R⁵ and R⁶ form asaturated, partially saturated or unsaturated, substituted orun-substituted bicyclic ring system, preferably a ten-membered bicyclicring containing one or more heteroatoms, preferably one or more nitrogenatoms, more preferably one nitrogen atom. Most preferably, the nitrogenatom of the heterocyclic ring corresponds to R⁵. It is further preferredthat the bicyclic ring system is saturated and/or unsubstituted.

According to a further preferred embodiment, R¹⁴ is a hydroxyl- and/orcarboxyl-substituted or unsubstituted alkyl. Examples for suchsubstituents are —(CH₂)₃—OH, —(CH₂)₅—COOH, and —CH₃.

According to a further preferred embodiment, X is a carbon atom. Thecarbon atom is preferably substituted, e.g. by one or two alkyl groupssuch as methyl or ethyl. Most preferably, X is —C(CH₃)₂.

According a further preferred embodiment, Z has the general formula IIa.

According a further preferred embodiment, n is 1.

According to a further preferred embodiment, R¹ is —C(CH₃)₃, R² ishydrogen, R³ is hydrogen, R⁴ is hydrogen, R⁵ is —N(CH₂CH₃)₂, R⁶ ishydrogen, R⁷ is hydrogen, R⁸ is hydrogen, R⁹ is hydrogen, R¹⁰ ishydrogen, R¹¹ is hydrogen, R¹² is hydrogen, R¹³ is hydrogen, R¹⁴ is—(CH₂)₃—OH, Z has the general formula IIa, X is —C(CH₃)₂ and/or n is 1.

According to a further preferred embodiment, R¹ is —C(CH₃)₃, R² ishydrogen, R³ is hydrogen, R⁴ is hydrogen, R⁵ is —NH₂, R⁶ is hydrogen, R⁷is hydrogen, R⁸ is hydrogen, R⁹ is hydrogen, R¹⁰ is hydrogen, R¹¹ ishydrogen, R¹² is hydrogen, R¹³ is hydrogen, R¹⁴ is —(CH₂)₃—OH, Z has thegeneral formula IIa, X is —C(CH₃)₂ and/or n is 1.

According to a further preferred embodiment, R¹ is —C(CH₃)₃, R² ishydrogen, R³ is hydrogen, R⁴ is hydrogen, R⁵ is —N(CH₂CH₃)₂, R⁶ ishydrogen, R⁷ is hydrogen, R⁸ is hydrogen, R⁹ is hydrogen, R¹⁰ ishydrogen, R¹¹ is hydrogen, R¹² is hydrogen, R¹³ is hydrogen, R¹⁴ is—CH₃, Z has the general formula IIa, X is —C(CH₃)₂ and/or n is 1.

According to a further preferred embodiment, R¹ is —C₆H₅, R² ishydrogen, R³ is hydrogen, R⁴ is hydrogen, R⁵ is —N(CH₂CH₃)₂, R⁶ ishydrogen, R⁷ is hydrogen, R⁸ is hydrogen, R⁹ is hydrogen, R¹⁰ ishydrogen, R¹¹ is hydrogen, R¹² is hydrogen, R¹³ is hydrogen, R¹⁴ is—(CH₂)₃—OH, Z has the general formula IIa, X is —C(CH₃)₂ and/or n is 1.

According to further preferred embodiments, the polymethine of thegeneral formula I is selected from one of the following compounds III toIX. Preferred counter-ions to the compounds having the general formula Iand especially to compounds III to IX are F⁻, Cl⁻, Br⁻, I⁻, ClO₄ ⁻ orBF₄ ⁻.

As used herein, the term “alkyl” means a linear or branched saturatedaliphatic hydrocarbon group having a single radical and 1-10 carbonatoms. Examples of alkyl groups include methyl, propyl, isopropyl,butyl, n-butyl, isobutyl, sec-butyl, tert-butyl and pentyl. A branchedalkyl means that one or more alkyl groups such as methyl, ethyl orpropyl, replace one or both hydrogens in a —CH₂ group of a linear alkylchain. The term “lower alkyl” means an alkyl of 1-3 carbon atoms.

The term “alkoxy” means an “alkyl” as defined above connected to anoxygen radical.

The term “cycloalkyl” means a non-aromatic mono- or multicyclichydrocarbon ring system having a single radical and 3-12 carbon atoms.Exemplary monocyclic cycloalkyl rings includes cyclopropyl, cyclopentyland cyclohexyl. Exemplary multicyclic cycloalkyl rings include adamantyland norbornyl.

The term “alkenyl” means a linear or branched aliphatic hydrocarbongroup containing a carbon-carbon double bond having a single radical and2-10 carbon atoms.

A “branched” alkenyl means that one or more alkyl groups such as methyl,ethyl or propyl replace one or both hydrogens in a —CH₂ or —CH═ linearalkenyl chain. Exemplary alkenyl groups include ethenyl, 1- and2-propenyl, 1-, 2- and 3-butenyl, 3-methylbut-2-enyl, 2-propenyl,heptenyl, octenyl and decenyl.

The term “cycloalkenyl” means a non-aromatic monocyclic or multicyclichydrocarbon ring system containing a carbon-carbon double bond having asingle radical and 3 to 12 carbon atoms. Exemplary monocycliccycloalkenyl rings include cyclopropenyl, cyclopentenyl, cyclohexenyl orcycloheptenyl. An exemplary multicyclic cycloalkenyl ring isnorbornenyl.

The term “aryl” means a carbocyclic aromatic ring system containing one,two or three rings which may be attached together in a pendent manner orfused, and containing a single radical. Exemplary aryl groups includephenyl, naphthyl and acenaphthyl.

The term “heterocyclic” or “heterocycle” means cyclic compounds havingone or more heteroatoms (atoms other than carbon) in the ring, andhaving a single radical. The ring may be saturated, partially saturatedor unsaturated, and the heteroatoms may be selected from the groupconsisting of nitrogen, sulfur and oxygen. Examples of saturatedheterocyclic radicals include saturated 3 to 6-memberedhetero-monocyclic groups containing 1 to 4 nitrogen atoms, such aspyrrolidinyl, imidazolidinyl, piperidino, piperazinyl; saturated 3- to6-membered hetero-monocyclic groups containing 1 to 2 oxygen atoms and 1to 3 nitrogen atoms such as morpholinyl; saturated 3- to 6-memberedhetero-monocyclic groups containing 1 to 2 sulfur atoms and 1 to 3nitrogen atoms, such as thiazolidinyl. Examples of partially saturatedheterocyclic radicals include dihydrothiophene, dihydropyran anddihydrofuran. Other heterocyclic groups can be 7 to 10 carbon ringssubstituted with heteroatoms such as oxocanyl and thiocanyl. When theheteroatom is sulfur, the sulfur can be a sulfur dioxide such asthiocanyldioxide.

The term “heteroaryl” means unsaturated heterocyclic radicals, wherein“heterocyclic” is as previously described. Exemplary heteroaryl groupsinclude unsaturated 3 to 6-membered hetero-monocyclic groups containing1 to 4 nitrogen atoms, such as pyrrolyl, pyridyl, pyrimidyl andpyrazinyl; unsaturated condensed heterocyclic groups containing 1 to 5nitrogen atoms, such as indolyl, quinolyl and isoquinolyl; unsaturated 3to 6-membered hetero-monocyclic groups containing an oxygen atom, suchas furyl; unsaturated 3 to 6-membered hetero-monocyclic groupscontaining a sulfur atom, such as thienyl; unsaturated 3 to 6-memberedhetero-monocyclic groups containing 1 to 2 oxygen atoms and 1 to 3nitrogen atoms, such as oxyzolyl; unsaturated condensed heterocyclicgroups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such asbenzoxazolyl; unsaturated 3 to 6-membered hetero-monocyclic groupcontaining 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, such asthiazolyl; and unsaturated condensed heterocyclic group containing 1 to2 sulfur atoms and 1 to 3 nitrogen atoms, such as benzothiazolyl. Theterm “heteroaryl” also includes unsaturated heterocyclic radicals,wherein “heterocyclic” is as previously described, in which theheterocyclic group is fused with an aryl group, in which aryl is aspreviously described. Exemplary fused radicals include benzofuran,benzdioxole and benzothiophene.

As used herein, the term “heterocyclic C₁₋₄ alkyl”, “heteroaromatic C₁₋₄alkyl” and the like refer to the ring structure bonded to a C₁₋₄ alkylradical,

As used herein, the term “ring” or “ring system” includes cycloalkyl,cycloalkenyl, aryl, heteroaryl or heterocycle.

All of the cyclic ring structures disclosed herein can be attached atany point where such connection is possible, as recognized by oneskilled in the art.

As used herein, the term “halogen” includes fluoride, bromide, chloride,or iodide.

BRIEF DESCRIPTION OF DRAWINGS

Other objects and many of the attendant advantages of embodiments of thepresent invention will be readily appreciated and become betterunderstood by reference to the following more detailed description ofembodiments in connection with the accompanied drawings.

FIG. 1 schematically illustrates the functional components required fora chip for utilization in the kit according to the present invention,illustrated in block diagram form.

FIG. 2 schematically illustrates an exemplary chip for utilization in akit according to the present invention.

FIG. 3 schematically illustrates a further exemplary chip forutilization in a kit according to the present invention.

FIG. 4 schematically illustrates another exemplary microscaleelectrophoresis device or chip for use in electrophoretic separation ofproteins such as lipoproteins for use in the present invention

FIG. 5 shows an electropherogram of a serum sample analyzed on amicrofluidic chip with the bioanalyzer according to example 1 (overlayof several electropherograms of the same sample run over an entirechip).

FIG. 6 shows the chemical formulae of exemplary marker dyes which aresuitable for use in a kit according to the present invention.

FIG. 7 shows a correlation between diameter and density of lipidparticles.

FIG. 8 shows the correlation of different lipoproteins with cardiacrisk.

The illustration in the drawings is schematically.

One component of the kit according to the present invention is a markerdye containing a polymethine with the general formula I. The substitutedderivatives of indole, heteroindole, pyridine, chinoline or acridine ofthe general formula I can be used as dyes for the optical marking orlabelling of proteins, in particular of lipoproteins.

The labelling of proteins can be done by the formation of ionic ornon-ionic interaction between the markers of general formula I and theproteins to be labelled. Alternatively, the functional groups of thesemarkers activated with regards to nucleophiles can couple covalentlywith an OH, NH₂ or SH function, which therefore creates a system for thequalitative and quantitative determination of proteins, in particular oflipoproteins.

The coupling or conjugation reaction can take place in an aqueous ormostly aqueous solution and preferably at room temperature. During thisreaction, a conjugate of a lipoprotein with the marker dye havingfluorescent properties is created.

By labelling lipoproteins with the above-described non-symmetricalpolymethines, which on the one hand have an easily derivatizableheterocycle of the type of the pyridine, chinoline, indole, heteroindoleor acridine derivatives, and on the other hand have a 6-ringheterocycle, in particular the following advantages are achieved:

In contrast to fluorescent dyes, which fluoresce independently of aformation of a conjugate with the proteins to be detected and thereforehave to be separated from the sample to be analysed before the detectionstep, the marker dyes according to the present invention haveessentially no fluorescent properties in an un-conjugated state.Further, the marker dyes according to the present invention selectivelyassociate with the unique micelle-like structure of lipoproteinscharacterized by hydrophobic residues in the interior and hydrophilicresidues on the exterior of the lipoprotein aggregates. Due tofluorescent properties of such a conjugate between a lipoprotein and themarker dye containing a polymethine having the general formula I aselective labelling of lipoproteins can be achieved.

By using the kits of the present invention, not only lipoproteins can beselectively detected but also HDL and LDL sub-class patterns oflipoproteins may be analysed.

As is apparent for the person skilled in the art, this advantage notonly holds true for lipoproteins, but for all kinds of micelle-likestructures such as proteins which are solubilized in a sufficient amountof SDS (sodium dodecyl sulphate), so that micelles are formed.

Further, trimethines according to the general formula I already absorbin the spectral range greater than 650 nm and have a significantlyimproved photochemical and thermal stability when compared to otherpolymethines known in the prior art.

By means of molecular engineering, it is possible to control theposition and intensity of the absorption and emission maxima at will andto adapt them to emission wavelengths of different excitation lasers, inparticular near infrared (NIR) laser diodes.

The marker dyes can be produced by a relatively simple two-stepsynthesis with which a variety of dyes with functionalities that differ,for example, with regards to the total charge of the dye and the number,specificity and reactivity of the activated group used for theimmobilization can be provided in a manner that is specific to therespective application. Polymethines having the general formula I aredescribed in U.S. Pat. No. 6,750,346 which is incorporated herein byreference in its entirety.

Compounds with the general formula I as well as systems derived fromthem i.e. conjugates of proteins such as lipoproteins and thepolymethines of the general formula I, can be used in optical, inparticular fluorescence optical qualitative and quantitativedetermination methods for electrophoresis, chromatography andelectrochromatography.

According to a preferred embodiment, the separation channel is adaptedfor separating different compounds electrophoretically,chromatographically or electrochromatographically. E.g., a chip forperforming an electrophoretic separation comprises a base substratecomprising a main surface, wherein a channel is formed in said mainsurface of said base substrate in at least one direction.

According to a further preferred embodiment, the kit further comprises aseparation gel. Examples of appropriate materials for inclusion in thisgel comprise polyacrylamide, polydimethylacrylamide, polyethylene oxideand/or polyvinylpyrrolidone. A preferred gel is a polydimethylacrylamide(PDMA) gel.

Optionally, the medium may also comprise a denaturing agent such asN-methylurea.

The chip may further comprise an element for applying an electric fieldacross the separation channel or the medium. The electric field isapplied across said separation channel by turning on a voltage. On thebasis of said separation mechanism a separation of the compounds in thesamples is performed.

In particular, the kit according to the present invention can beemployed in various electrophoretic techniques. Non-limiting examples ofelectrophoretic techniques include SDS polyacrylamide gelelectrophoresis (SDS-PAGE), capillary electrophoresis, andmicro-channel/microfluidic channel electrophoresis.

A preferred type of electrophoresis to employ the kit ismicrofluidic-channel electrophoresis.

Another component of the kit according to the present invention whichmay also be used in the methods according to the present invention is achip for performing a separation of macromolecular species such asproteins and in particular lipoproteins.

If the kit is used for microfluidic-channel electrophoresis, itpreferably involves a micro-channel chip having a network ofmicro-channels that serve as paths for the migration of fluid samplevolumes. A single sample volume or many sample volumes may be run on thesame micro-channel chip simultaneously. The micro-channel chip is loadedinto a device, such as a bioanalyzer for molecular assays (e.g., anAgilent 2100 bioanalyzer), which provides a network of microelectrodesonto the chip wells, thus supplying the necessary voltages and currentsfor the separation of the sample volume components. Micro-channel chipelectrophoresis generally provides higher resolutions, smaller samplevolume sizes, shorter analysis times, and reduced sample handling overtraditional capillary electrophoresis. An example of this type ofelectrophoresis is described in U.S. Pat. No. 6,042,710, which is herebyincorporated herein by reference in its entirely.

When used for microfluidic-channel electrophoresis, the chip can haveelectrodes and a substrate which comprises a planar body structure inwhich grooves are fabricated to define capillary channels when overlaidwith a cover element, also typically planar in structure. Exemplarysubstrates materials include, e.g. glass, quartz, silica, silicon,polymers, e.g. plastics like polydimethylsiloxanes (PDMS),polymethylmethacrylate (PMMA), polyurethane, polyvinylchloride (PVC),polystyrene, polysulfone, polycarbonate, polytetrafluoroethylene(Teflon™), and a variety of others that are well known in the art.Substrates may take a variety of shapes or forms, including tubularsubstrates, e.g. polymer or fused silica capillaries, or the like. Inpreferred aspects, however, the substrate comprises a planar bodystructure in which grooves are fabricated to define capillary channelswhen overlaid with a cover element, also typically planar in structure.Examples of such planar capillary systems are described in U.S. Pat. No.5,976,336 and are incorporated herein by reference in its entirety. Themedium is employed in the micro-channels formed in the substrate tobring about the separation of sample components passing through themicro-channels under the influence of an electric field induced acrossthe medium by the electrodes.

Capillary channels also can be of a variety of different shapes incross-section, including tubular channels, rectangular channels,rhomboid channels, hemispherical channels or the like, or even morearbitrary shapes such as may result from less precise fabricationtechniques, e.g. laser ablation. Typically, the shape of a capillarychannel will vary, depending upon the substrate type used and the methodof propagation. For example, in typical fused silica capillaries, thecapillary channel will be tubular. In systems employing planarsubstrates, channels will typically comprise either a rhomboid,rectangular or hemispherical cross sectional shape, depending upon thesubstrate material and method of fabrication of the channels.

A variety of manufacturing techniques are well known in the art forproducing microfabricated channel systems. For example, where suchdevices utilize substrates commonly found in the semiconductor industry,manufacturing methods regularly employed in those industries are readilyapplicable, e.g. photolithography, wet chemical etching, chemical vapourdeposition, sputtering, electroforming, etc. Similarly, methods offabricating such devices in polymeric substrates are also readilyavailable including injection molding, embossing, laser ablation, LIGAtechniques and the like. Other useful fabrication techniques includelamination or layering techniques, used to provide intermediatemicro-scale structures to define elements of a particular micro-scaledevice.

Typically, the capillary channels will have an internal cross-sectionaldimension, e.g. width, depth, or diameter, of between about 1 μm andabout 500 μm, with most such channels having a cross-sectional dimensionin the range of from about 10 μm to about 200 μm.

In particular, the preferred aspects, planar micro-fabricated devicesemploying multiple integrated micro-scale capillary channels are used.Briefly, these planar micro-scale devices employ an integrated channelnetwork fabricated into the surface of a planar substrate. A secondsubstrate is overlaid on the surface of the first to cover and seal thechannels, and thereby define the capillary channels.

Preferably, the chip of the kit according to the present invention isprovided with one or more analysis channels or separation channels orseparation flow paths and comprises additional channels connecting theanalysis channel to multiple different sample reservoirs. Thesereservoirs are generally defined by apertures disposed in the secondoverlaying substrate, and positioned such that they are in fluidcommunication with the channels of the device. A variety of specificchannel geometries are employed to optimise channel layout in terms ofmaterial transport time, channel lengths and substrate use. Examples ofsuch micro-scale channel network systems are described in detail in WO98/49548, U.S. Pat. No. 6,235,175, U.S. Pat. No. 6,153,073, U.S. Pat.No. 6,068,752, U.S. Pat. No. 5,976,336 and U.S. application Ser. No.60/060,902, which are all incorporated herein by reference in itsentirety.

Introduction of the separation gel or medium into a capillary channel ormicro-channel may be as simple as placing one end of the channel intocontact with the medium and allowing the medium to wick into thechannel. Alternatively, vacuum or pressure may be used to drive themedium solution into the capillary channel. In integrated channelsystems such as those used in chip electrophoresis, the medium istypically placed into contact with a terminus of a common micro-channel,e.g. a reservoir disposed at the end of a separation channel, and slightpressure is applied to force the polymer into all of the integratedchannels.

A preferred method according to the present invention, in particular ifthe separation is performed electrophoretically, comprises the followingsteps:

-   -   injecting the sample into a chip, wherein the chip comprises at        least one well for receiving the sample, and a separation        channel coupled to the at least one well and being adapted for        separating different compounds; and    -   applying an electric field across the channel to move the sample        through the channel.

A sample containing proteins or lipoproteins for which separation isdesired is preferably placed in one end of the separation channel and avoltage gradient is applied along the length of the channel. As thesample components are electrokinetically transported down the length ofthe channel and through the medium disposed therein, those componentsare resolved. The separated components are then detected at a pointalong the length of the channel, typically near the terminus of theseparation channel distal to the point at which the sample wasintroduced.

The separation in the method according to the present invention ispreferably performed at a pH in the range of from about 7 to about 8,more preferably at a pH in the range of from about 7.3 to about 7.7 andmost preferred at pH of about 7.5.

Further, it is preferred that the sample containing proteins orlipoproteins for which separation is desired comprises sodium dodecylsulphate, preferably in an amount of from about 0.10 to about 0.20 mM,more preferably in an amount of from about 0.125 to about 0.175 mM andmost preferred in an amount of about 0.15 mM.

The marker according to the general formula I dye may be injected intothe chip together with the sample to be analyzed, or before or after thesample has been injected. Alternatively or in addition, the marker dyeaccording to the general formula I may be contained in the separationgel. If the marker dye according to the general formula I is present inthe separation gel it may serve to focus the detection device.

Detection of separated species is typically carried out using afluorescent detection system that is well known in the art. Typically,such detection systems operate by detecting fluorescence of the markerdye which contains a polymethine of the general formula I as describedabove. Typically, such systems utilize a light source capable ofdirecting light energy at the separation channel as the separatedspecies are transported past. The light source typically produces lightof an appropriate wavelength to activate the labelling group.Fluorescent light from the labelling group is then collected byappropriate optics, e.g. an objective lens, located above, below oradjacent the capillary channel, and the collected light is directed at aphotometric detector, such as a photodiode or photomultiplier tube. Thedetector is typically coupled to a computer, which receives the datafrom the detector and records that data for subsequent storage andanalysis.

Before a sample comprising a plurality of unknown species is analysed,the measurement set-up is usually calibrated. Hence, it is preferred,that the kit further comprises a calibration sample. The calibrationsample can be selected from a large variety of different calibrationsamples comprising a set of well-known compounds of different size suchas SRM 1951b—Lipids in Frozen Human, Serum, Level 1 (NIST, Gaithersburg,Md., USA), Ultra HDL calibrator vial, 1 ml (Genzyme Diagnostics, WestMalling Kent, ME, UK), Human HDL, 10 mg; Human LDL, 5 mg; Human Ox. LDL,2 mg; Human Lp(a), 0.1 mg (all available at BTI, BiomedicalTechnologies, Inc., MA, USA), AutoHDL/LDL Calibrator, 3 ml; HDLStandard, 15 ml (both available at Eco-Scientific, Rope Walk, Thrupp,Stroud, UK), Lipid Control Levels 1, 2 and 3 (all available atPolymedco, Inc., Cortland Manor, N.Y., USA), Low total cholesterol, TCh@ 50 mg/dL, LRC LEVEL 1; Normal total cholesterol, TCh @ 165-180 mg/dL,TG<100 mg/dL, LRC LEVEL 2; Elevated total cholesterol, TCh @ 265, TG @230; LRC LEVEL 3; High Density Lipoprotein, HDL @ 50, LRC LEVEL 4 (allavailable at Solomon Park Research Laboratories, Kirkland, Wash., USA),and HDL Reference Pools ID 204 (TV (SD) 60.1 (0.7) mg/dL), ID 205 (TV(SD) 30.5 (0.8) mg/dL), ID 301 (TV (SD) 49.5 (1.2) mg/dL), ID 303 (TV(SD) 50.6 (1.4) mg/dL), ID 305 (TV (SD) 30.8 (0.8) mg/dL), ID 307 (TV(SD) 40.5 (0.9) mg/dL) (all available at Centers for Disease Control andPrevention Atlanta, Ga. 3034, USA; note: pools may be prepared accordingto the Lipid Standardization Program (LSP)).

It is particularly preferred that a so-called ladder is used as acalibration sample. A ladder is a calibration sample comprising aplurality of well-known components, whereby the name “ladder” is due tothe fact that the calibration peak pattern looks like a ladder of peaksrelated to the various components. Because the set of calibration peakslooks like a ladder, calibration samples are often referred to as“ladders”. A lot of manufacturers in the field of DNA analysis andprotein analysis produce calibration samples or “ladders” forelectrophoresis systems, chromatography systems or electrochromatographysystems. E.g., in protein analysis, calibration samples comprising a setof different proteins are used.

In case fluorescence detection is used for detecting different species,ladders comprising fragments labelled with fluorescence tags may beemployed. When the species of the calibration sample are stimulated withincident light, the tags attached to the species emit fluorescencelight. Calibration samples or “ladders” comprising a marker thatfluoresces at a first wavelength, and a set of labelled fragments thatemit fluorescent light at a second wavelength may also be employed.

After the fluorescent peak pattern of the calibration sample has beenacquired, a sample of interest is analysed. In order to allow for analignment with the calibration peak pattern, a certain concentration ofthe marker and a certain concentration of the largest labelled ladderfragment may be added to the sample of interest. Then, the compounds ofthe sample of interest are separated, and the samples bands obtained atthe separation column's outlet are analysed.

According to another preferred embodiment, the marker dye according toformula I emits fluorescent light of a first wavelength, whereas thelabelled fragments of the calibration sample emit fluorescence light ofa second wavelength, which is different from the first wavelength. Someof the available ladders comprise two or more different fluorescencedyes adapted for emitting fluorescence light of two or more differentwavelengths. Correspondingly, there exist fluorescence detection unitsadapted for simultaneously tracking fluorescence intensity at two ormore wavelengths.

Preferred methods for peak pattern calibration are disclosed in Europeanpatent application 1 600 771 which is incorporated herein by referencein its entirety.

In the following, a preferred embodiment of the method of analysingproteins according to the present invention in described.

Initially, in the area of material flow, the materials to be examined,possibly in addition to the reagents required for the corresponding testsuch as the marker dyes having the general formula I, are fed to themicrochip. Thereafter, these materials on the microchip are moved ortransported, e.g. by means of electrical forces, pressure sources,thermal sources or the like. The feed and/or the movement of materialsmay be brought about by means of a suitable electronic control.

In this example, the materials are subjected to preliminary treatmentbefore they undergo the test as such. This preliminary treatment may,for example, consist of preheating by means of a heating system orpre-cooling by means of a suitable cooling system, in order, forexample, to fulfil the required thermal test conditions. As is known,the temperature conditions for execution of a chemical test usuallyexert a considerable influence on the cycle of test kinetics. Such apreliminary treatment can also take place in a multiple sequence, inwhich context a pre-treatment cycle and a further transport cycle areobviated. The above-mentioned pre-treatment can in this instance, inparticular, fulfil the function of separation of materials such as toaccess only certain specified components of the initial materials forthe corresponding test. Essentially, both the material quantity(quantity) and the material speed (quality) can be determined by meansof the transportation as described. In particular, precise adjustment ofmaterial quantity enables precise metering of individual materials andmaterial components. Further, the latter processes can advantageously becontrolled by means of electronic control.

After one or more optional pre-treatments, the actual experimentaltest/examination takes place, in which context the test results can bedetected on a suitable detection point of the chip or microchip.Detection advantageously takes place by means of optical detection, e.g.by a laser diode in conjunction with a photoelectric cell, or a massspectrometer, which may be connected or by means of electricaldetection. The resultant optical measurement signals are then fed to asignal-processing system and thereafter to an analysis unit (e.g. asuitable microprocessor) for interpretation of the measurement results.

The operational components typically used for a chip contained in thekit according to present invention described herein are schematicallyillustrated in FIG. 1. These are mainly subdivided into the componentsrelating to a material transport or flow 1, and those which representthe information flow 2 arising upon execution of a test. Material flow 1typically includes sampling operations 3 and operations for transporting4 materials on the chip, as well as optional operations for treatment orpre-treatment 5 of the materials to be examined. Furthermore, a sensorsystem 6 is typically employed to detect the results of a test and,optionally, to monitor the material flow operations, so that adjustmentscan be made in controlling material flow using the control system. Oneexample of the control mechanism is shown as control electronics 7. Dataobtained in the detection operation 6 and 6′ is transferred typically tothe signal processing 8 operation so that the detected measurementresults can be analysed. A priority objective in the design of suchmicrochip systems is the provision of function units/modulescorresponding to the above-mentioned functions and the establishment ofsuitable interfaces between individual modules. By means of a suitabledefinition of these interfaces, it is possible to achieve a high degreeof flexibility in adapting the systems to various microchips orexperimental arrangements. Furthermore, on the basis of such a strictlymodular system structure, it is possible to achieve the most extensivelevel of compatibility between various microchips and/or microchipsystems.

Initially, in the area of material flow, the materials to be examined(possibly in addition to the reagents required for the correspondingtest) are fed to the microchip 3. Thereafter, these materials on themicrochip are moved or transported, e.g., by means of electrical forces4. Both the feed and the movement of materials are brought about bymeans of a suitable electronic control 7, as indicated by means of thedotted line. In this example, the materials are subjected to preliminarytreatment 5, before they undergo the test as such. This preliminarytreatment may, for example, consist of pre-heating by means of a heatingsystem or pre-cooling by means of a suitable cooling system in order,for example, to fulfill the required thermal test conditions. As isknown, the temperature conditions for execution of a chemical testusually exert a considerable influence on the cycle of test kinetics. Asis indicated by the arrow, this preliminary treatment can also takeplace in a multiple sequence, in which context there are obviated apretreatment cycle 5 and a further transport cycle 4′. Theabove-mentioned pretreatment can in this instance, in particular,fulfill the function of separation of materials such as to access onlycertain specified components of the initial materials for thecorresponding test. Essentially, both the material quantity (quantity)and the material speed (quality) can be determined by means of thetransportation as described. In particular, precise adjustment ofmaterial quantity enables precise metering of individual materials andmaterial components. Furthermore, the latter processes canadvantageously be controlled by means of electronic control 7.

After one or more pre-treatments, the actual experimentaltest/examination takes place, in which context the test results can bedetected on a suitable detection point of the microchip 6. Detectionadvantageously takes place by means of optical detection, e.g. a laserdiode in conjunction with a photoelectric cell, a mass spectrometer,which may be connected, or by means of electrical detection. Theresultant optical measurement signals are then fed to asignal-processing system 8, and thereafter to an analysis unit (e.g.suitable microprocessor) for interpretation 9 of the measurementresults.

Following the above-mentioned detection 6, there is the option ofimplementation, as indicated by the dotted line, of further test seriesor analyses or separation of materials, e.g., those in connection withvarious test stages of a chemical test cycle which is, overall, morecomplicated. For this purpose, materials are transported onwards on themicrochip after the first detection point 6, and to a further detectionpoint 6′. There, the procedure theoretically defined according to stages4′ and 6 is performed. Finally, the materials are fed, after terminationof all reactions/tests, to a material drain (not illustrated here) bymeans of a concluding transport cycle or collection cycle 4′″.

Further incentives for miniaturizations in the field of chemicalanalysis include the ability and desirability to minimize the distanceand time over which materials are transported. In particular, the amountof time and distance required to transport materials between thesampling of the materials and the respective detection point of anychemical reaction that has taken place shall be minimized. It isfurthermore known from the field of liquid chromatography andelectrophoresis that separation of materials can be achieved morerapidly and individual components can be separated with a higher degreeof resolution than has been possible in conventional systems.Furthermore, micro-miniaturized laboratory systems enable a considerablyreduced consumption of materials, particularly reagents, and a far moreefficient intermixing of the components of materials. A preferredapparatus for the operation of a microfluidic device, i.e. a microchiplaboratory system for chemical processing or analysis, is described inWO 00/78454 which is incorporated herein by reference in its entirety.

FIG. 2 shows an exemplary laboratory microchip or chip which is suitablefor utilization in a kit or method according to the invention. On theupper side of a substrate 20, microfluidic structures are provided,through which materials are transported. Substrate 20 may, for example,be made up of glass or silicon, in which context the structures may beproduced by means of a chemical etching process or a laser etchingprocess. Alternatively, such substrates may include polymeric materialsand be fabricated using known processes such as injection molding,embossing and laser ablation techniques. Typically, such substrates areoverlaid with additional substrates in order to seal the conduits asenclosed channels or conduits.

For sampling of the material to be examined (hereinafter called the“sample material”) onto the microchip, one or several recesses 21 areprovided on the microchip, to function as reservoirs for the samplematerial. In performing a particular exemplary analysis or test, thesample material is initially transported along a transport duct orchannel 25 on the microchip. In this example, transport channel 25 isillustrated as a V-shaped groove for convenience of illustration.However, the microfluidic substrates typically comprise sealedrectangular (or substantially rectangular) or circular-section conduitsor channels.

The reagents required for the test cycle are typically accommodated inrecesses 22 which also fulfil the function of reagent and/or samplematerial reservoirs. In this example, two different materials couldreadily be manipulated. By means of corresponding transport conduits 26,these are initially fed to a junction point 27, where they intermix and,after any chemical analysis or synthesis has been completed, constitutethe product ready to use. At a further junction 28 this reagent meetsthe material sample to be examined, in which the two materials will alsointermix.

The material formed then passes through a conduit section 29 which mayhave a meandering geometry which functions to achieve artificialextension of the distance available for reaction between the materialspecimen and the reagent. In a further recess 23 configured as amaterial reservoir, in this example, there is contained a furtherreagent which is fed to the already available material mix at a furtherjunction point 31.

The reaction of interest takes place after the above-mentioned junctionpoint 31, which reaction can then be detected, ideally by contactlessmeans, e.g. optically, within an area 32 (or measurement zone) of thetransport duct by means of a detector. In this context, thecorresponding detector can be located above or below area 32. After thematerial has passed through the above-mentioned area 32, it is fed to afurther recess 24 which represents a waste reservoir or material drainfor the waste materials which have been produced, overall, in the courseof the reaction.

Finally, on the microchip there are provided recesses 33 which act ascontactless surfaces for application of electrodes and which in turnenable the electrical voltages, and even high voltages, required forconnection to the microchip for operation of the chip. Alternatively,the contacting for the chips can also take place by means of insertionof a corresponding electrode point directly into the recesses 21, 22, 23and 24 provided as material reservoirs. By means of a suitablearrangement of electrodes 33 along transport conduits 25, 26, 29 and 30and a corresponding chronological or intensity-related harmonization ofthe applied fields, it is then possible to achieve a situation in whichthe transportation of individual materials takes place according to aprecisely dictated time/quantity profile, such that it is possible toachieve very precise consideration of and adherence to the kinetics forthe underlying reaction process.

In pressure-driven transport of materials within the microfluidicstructure, it is typically necessary to make recesses 33 such thatcorresponding pressure supply conduits closely and sealably engage themso as to make it possible to introduce a pressurized medium, for examplein inert gas, into the transport conduits, or apply an appropriatenegative pressure.

FIG. 3 shows a further exemplary measurement set-up for separating andanalysing a fluid sample comprising a plurality of different samplecompounds. Each of the sample compounds is characterized by anindividual migration time required for travelling through a separationflow path 1. The separation flow path 1 might e.g. be an electrophoresisflow path, a chromatography flow path or an electric chromatography flowpath. At the outlet of the separation flow path, a detection cell islocated. The detection cell might e.g. be implemented as a fluorescencedetection cell 2 comprising a light source 3 and a fluorescencedetection unit 4. The fluorescence detection cell 2 is adapted fordetecting sample bands of fluorescence-labelled species as a function oftime.

FIG. 4 shows a further specific example of channel geometry of a chip ina kit according to the present invention. In operation, sample materialsare placed into one or more of the sample reservoirs 116-138. A firstsample material, e.g., disposed in reservoir 116, is then loaded byelectrokinetically transporting it through channels 140 and 112, andacross the intersection with the separation channel 104, towardload/waste reservoir 186 through channel 184. Sample is then injected bydirecting electrokinetic flow from buffer reservoir 106 through analysischannel 104 to waste reservoir 108, while pulling back the sample in theloading channels 112:114 at the intersection. While the first sample isbeing separated in analysis channel 104, a second sample, e.g., thatdisposed in reservoir 118, is preloaded by electrokineticallytransporting it into channels 142 and 112 and toward the load wastereservoir 184 through channel 182. After separation of the first sample,the second sample is then loaded across the intersection with analysischannel 104 by transporting the material towards load/waste reservoir186 through channel 184.

Further methods of electrophoretically separating macromolecular speciessuch as proteins, as well as compositions, systems, devices or chipsuseful in carrying out such methods are described in U.S. Pat. No.6,042,710 which is incorporated herein by reference in its entirety.

Other preferred devices for operating a microchip with a microfluidstructure for chemical, physical and/or biological processing aredescribed in European patent application 1 360 992 and internationalpatent application WO 00/78454 which are both also incorporated hereinby reference in its entirety.

All publications and patent applications are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference in its entirety. Although the presentinvention has been described in some detail by way of illustration andexample for purposes of clarity and understanding, it will be apparentthat certain changes and modifications may be practised within the scopeof the dependent claims.

It should be noted that the term “comprising” does not exclude otherelements or features and the “a” or “an” does not exclude a plurality.Also elements described in association with different embodiments may becombined. It should also be noted that reference signs in the claimsshall not be construed as limiting the scope of the claims.

The invention is subsequently to be illustrated in more detail by meansof an embodiment example.

EXAMPLE

In the following a typical application of the kit according to thepresent invention for the separation of lipoproteins is shown:

The sample buffer contains the following reagents

-   -   200 mM TAPS(N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic        acid), pH 7.5 (Sigma, Deisenhofen, Germany)    -   6 μM dye V02-04064 (Dyomics, Jena, Deutschland) as the staining        marker dye    -   1 μM Alexa 700 (Invitrogen—Molecular Probes, USA) as the lower        marker for calibration    -   1 μM dye 676 (Dyomics) as the upper marker for calibration    -   0.15 mM SDS (sodium dodecyl sulfate) (Sigma)

The separation gel or gel matrix contains the following reagents:

-   -   2% PDMA 12164 (Polysciences; USA; lot No.: 537426)    -   200 mM TAPS(N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic        acid), pH 7.5 (Sigma)    -   0.15 mM SDS (Sigma)    -   0.15 μM dye V02-04064 (Dyomics) as the dye for focusing the        detector

The following assay protocol was used:

-   -   Take 1 μL of 12 different human serum samples and mix with 49 μL        each of sample buffer containing dye.    -   Take 1 μL of serum standard and mix with 49 μl each of sample        buffer containing dye.    -   Place chip on primer station.    -   Label each chip.    -   Add 10 μL of 2% gel matrix to the gel well.    -   Pressurize the well for 1 min.    -   Fill the other two gel wells with 10 μL gel matrix.    -   Add 10 μl of diluted serum standard in sample buffer to the        standard well    -   Add 7 μL of each diluted sample in sample buffer to each of the        12 sample wells.    -   Place chip into instrument and start run.

For performing the assay, the Agilent 2100 Bioanalyzer (AgilentTechnologies, USA) was used.

A typical electropherogram of a serum sample analyzed on a microfluidicchip with the bioanalyzer is shown in FIG. 5.

1. A kit for optically detecting proteins, in particular lipoproteins,in a sample, the kit comprising: a chip for performing a separation ofthe proteins, in particular of the lipoproteins, wherein the chipcomprises at least one well for receiving the sample, and a separationchannel coupled to the at least one well and being adapted forseparating different compounds and a marker dye which contains apolymethine of the general formula I

wherein Z is a substituted derivative of benzooxazole, benzodiazole,2,3,3-trimethylindolinine, 2,3,3-trimethyl-4,5-benzo-3H-indolinine, 3-and 4-picoline, lipidine, chinadine and 9-methylacridine derivativeswith the general formula IIa or IIb or IIc

and wherein X is selected from the group consisting of O, S, Si, N-alkyland C(alkyl)₂, n is 0, 1, 2 or 3, R¹ to R¹⁴ are independently selectedfrom the group consisting of hydrogen, alkyl, alkoxy, cycloalkyl, linearor branched alkenyl, cycloalkenyl, aryl, heteroaryl, heterocycle,hydroxy, carboxy, amine, alkyl-substituted amine and cyclic amine and/ortwo or more fragments in ortho-position to each other, for example R¹⁰and R¹¹ or R⁴, R⁵ and R⁶ together form another cycloalkyl ring or ringsystem, heterocyclic ring or ring system, heteroaryl ring system oraromatic ring or ring system.
 2. The kit of claim 1, wherein theseparation channel is adapted for separating different compoundselectrophoretically, chromatographically or electrochromatographically.3. The kit of claim 2, wherein the separation channel is adapted forseparating different compounds electrophoretically by electrophoresisselected from the group consisting of SDS polyacrylamide electrophoresis(SDS-PAGE), capillary electrophoresis and micro-channel/microfluidicchannel electrophoresis.
 4. The kit of claim 1 or any of the aboveclaims, wherein the chip further comprises an element for applying anelectrical field across the separation channel.
 5. The kit of claim 1 orany of the above claims, wherein the chip comprises a material selectedfrom the group consisting of glass, quartz, silica, silicon, andpolymers.
 6. The kit of claim 1 or any of the above claims, furthercomprising a separation gel.
 7. The kit of claim 6, wherein theseparation gel is selected from the group consisting of polyacrylamide,polydimethylacrylamide, polyethylene oxide, and polyvinyl pyrrolidoneand is preferably polydimethylacrylamide.
 8. The kit of claim 1 or anyof the above claims, further comprising a calibration sample.
 9. The kitaccording to claim 8, wherein the calibration sample is a “ladder”. 10.The kit of claim 1 or any of the above claims, wherein R¹ is —(CH₃)₃.11. The kit of claim 1 or any of the above claims, wherein R², R³, R⁴,R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and/or R¹³ is hydrogen.
 12. The kit ofclaim 1 or any of the above claims, wherein R⁵ is —N(CH₂CH₃)₂.
 13. Thekit of claim 1 or any of the above claims, wherein R¹⁴ is selected formthe group consisting of —(CH₂)₃—OH, —(CH₂)₅—COOH, and —CH₃.
 14. The kitof claim 1 or any of the above claims, wherein X is —C(CH₃)₂.
 15. Thekit of claim 1 or any of the above claims, wherein Z has the generalformula IIa.
 16. The kit of claim 1 or any of the above claims, whereinn is
 1. 17. The kit of claim 1 or any of the above claims, wherein R¹ is—C(CH₃)₃, R² is hydrogen, R³ is hydrogen, R⁴ is hydrogen, R⁵ is—N(CH₂CH₃)₂, R⁶ is hydrogen, R⁷ is hydrogen, R⁸ is hydrogen, R⁹ ishydrogen, R¹⁰ is hydrogen, R¹¹ is hydrogen, R¹² is hydrogen, R¹³ ishydrogen, R¹⁴ is —(CH₂)₃—OH, Z has the general formula IIa, X is—C(CH₃)₂ and/or n is
 1. 18. The kit of claim 1 or any of the aboveclaims, wherein R¹ is —C(CH₃)₃, R² is hydrogen, R³ is hydrogen, R⁴ ishydrogen, R⁵ is —NH₂, R⁶ is hydrogen, R⁷ is hydrogen, R⁸ is hydrogen, R⁹is hydrogen, R¹⁰ is hydrogen, R¹¹ is hydrogen, R¹² is hydrogen, R¹³ ishydrogen, R¹⁴ is —(CH₂)₃—OH, Z has the general formula IIa, X is—C(CH₃)₂ and/or n is
 1. 19. The kit of claim 1 or any of the aboveclaims, wherein R¹ is —C(CH₃)₃, R² is hydrogen, R³ is hydrogen, R⁴ ishydrogen, R⁵ is —N(CH₂CH₃)₂, R⁶ is hydrogen, R⁷ is hydrogen, R⁸ ishydrogen, R⁹ is hydrogen, R¹⁰ is hydrogen, R¹¹ is hydrogen, R¹² ishydrogen, R¹³ is hydrogen, R¹⁴ is —CH₃, Z has the general formula IIa, Xis —C(CH₃)₂ and/or n is
 1. 20. The kit of claim 1 or any of the aboveclaims, wherein R¹ is —C₆H₅, R² is hydrogen, R³ is hydrogen, R⁴ ishydrogen, R⁵ is —N(CH₂CH₃)₂, R⁶ is hydrogen, R⁷ is hydrogen, R⁸ ishydrogen, R⁹ is hydrogen, R¹⁰ is hydrogen, R¹¹ is hydrogen, R¹² ishydrogen, R¹³ is hydrogen, R¹⁴ is —(CH₂)₃—OH, Z has the general formulaIIa, X is —C(CH₃)₂ and/or n is
 1. 21. The kit of claim 1 or any of theabove claims, wherein the polymethine of the general formula I isselected from one of the following compounds III to IX:


22. A method of analysing lipoproteins in a sample, the methodcomprising: performing a separation of the lipoproteins in at least onedimension; labelling the lipoproteins, with a marker dye containing apolymethine of the general formula I

wherein Z is a substituted derivative of benzooxazole, benzodiazole,2,3,3-trimethylindolinine, 2,3,3-trimethyl-4,5-benzo-3H-indolinine, 3-and 4-picoline, lipidine, chinadine and 9-methylacridine derivativeswith the general formula IIa or IIb or IIc

and wherein X is selected from the group consisting of O, S, Si, N-alkyland C(alkyl)₂, n is 0, 1, 2 or 3, R¹ to R¹⁴ are independently selectedfrom the group consisting of hydrogen, alkyl, alkoxy, cycloalkyl, linearor branched alkenyl, cycloalkenyl, aryl, heteroaryl, heterocycle,hydroxy, carboxy, amine, alkyl-substituted amine and cyclic amine and/ortwo or more fragments in ortho-position to each other, for example R¹⁰and R¹¹ or R⁴, R⁵ and R⁶ together form another cycloalkyl ring or ringsystem, heterocyclic ring or ring system, heteroaryl ring system oraromatic ring or ring system; and optically detecting the separated andlabelled lipoproteins.
 23. The method of claim 22, wherein theseparation is performed electrophoretically, chromatographically orelectrochromatograpically.
 24. The method of claim 23, wherein theelectrophoretic separation is selected from the group consisting of SDSpolyacrylamide electrophoresis (SDS-PAGE), capillary electrophoresis andmicro-channel/microfluidic channel electrophoresis.
 25. The method ofclaim 22 or any of claims 23 or 24, wherein performing theelectrophoretic separation comprises: injecting the sample into a chip,wherein the chip comprises at least one well for receiving the sample,and a separation channel coupled to the at least one well and beingadapted for separating different compounds; applying an electric fieldacross the channel to move the sample through the channel.
 26. Themethod of claim 22 or any of claims 23 to 25, wherein the separation isperformed on a separation gel selected from the group consisting ofpolyacrylamide, polydimethylacrylamide, polyethylene oxide, andpolyvinyl pyrrolidone, and is preferably performed on apolydimethylacrylamide gel.
 27. The method of claim 25 or 26, whereinthe chip comprises a material selected from the group consisting ofglass, quartz, silica, silicon, and polymers.
 28. The method of claim 22or any of claims 23 to 27, wherein the separation is performed at a pHin the range of from about 7 to about 8, preferably at a pH in the rangeof from about 7.3 to about 7.7 and more preferred at pH of about 7.5.29. The method of claim 22 or any of claims 23 to 28, wherein the samplecomprises sodium dodecyl sulphate, preferably in an amount of from about0.10 to about 0.20 mM, more preferably in an amount of from about 0.125to about 0.175 mM and mot preferred in an amount of about 0.15 mM. 30.The method of claim 22 or any of claims 23 to 29, wherein a calibrationis performed before analysis.
 31. The method according to claim 28,wherein the calibration is performed with a “ladder”.
 32. The method ofclaim 22 or any of claims 23 to 30, wherein the separated and labelledlipoproteins are optically detected by fluorescence spectroscopy. 33.The method of claim 22 or any of claims 23 to 32, wherein the method isperformed by using a kit according to claim 1 or any of claims 2 to 21.34. A method of optically detecting lipoproteins in a sample, whereinthe method comprises labelling the lipoproteins, with a marker dyecontaining a polymethine of the general formula I

wherein Z is a substituted derivative of benzooxazole, benzodiazole,2,3,3-trimethylindolinine, 2,3,3-trimethyl-4,5-benzo-3H-indolinine, 3-and 4-picoline, lipidine, chinadine and 9-methylacridine derivativeswith the general formula IIa or IIb or IIc

and wherein X is selected from the group consisting of O, S, Si, N-alkyland C(alkyl)₂, n is 0, 1, 2 or 3, R¹ to R¹⁴ are independently selectedfrom the group consisting of hydrogen, alkyl, alkoxy, cycloalkyl, linearor branched alkenyl, cycloalkenyl, aryl, heteroaryl, heterocycle,hydroxy, carboxy, amine, alkyl-substituted amine and cyclic amine and/ortwo or more fragments in ortho-position to each other, for example R¹⁰and R¹¹ or R⁴, R⁵ and R⁶, together form another cycloalkyl ring or ringsystem, heterocyclic ring or ring system, heteroaryl ring system oraromatic ring or ring system; and optically detecting the labelledlipoproteins.
 35. The method of claim 34, wherein the method isperformed by using a marker dye as defined in any of claims 10 to 21.36. Use of a polymethine of the general formula (I) for opticallydetecting lipoproteins in a sample by labelling the lipoproteins withthe polymethine of the general formula I

wherein Z is a substituted derivative of benzooxazole, benzodiazole,2,3,3-trimethylindolinine, 2,3,3-trimethyl-4,5-benzo-3H-indolinine, 3-and 4-picoline, lipidine, chinadine and 9-methylacridine derivativeswith the general formula IIa or IIb or IIc

and wherein X is selected from the group consisting of O, S, Si, N-alkyland C(alkyl)₂, n is 0, 1, 2 or 3, R¹ to R¹⁴ are independently selectedfrom the group consisting of hydrogen, alkyl, alkoxy, cycloalkyl, linearor branched alkenyl, cycloalkenyl, aryl, heteroaryl, heterocycle,hydroxy, carboxy, amine, alkyl-substituted amine and cyclic amine and/ortwo or more fragments in ortho-position to each other, for example R¹⁰and R¹¹ or R⁴, R⁵ and R⁶, together form another cycloalkyl ring or ringsystem, heterocyclic ring or ring system, heteroaryl ring system oraromatic ring or ring system.
 37. The use of claim 36, wherein themarker dye is defined as in any of claims 10 to
 21. 38. Use of apolymethine of the general formula (I) for the analysis of lipoproteinclass distribution in a sample by labelling lipoproteins with thepolymethine of the general formula I

wherein Z is a substituted derivative of benzooxazole, benzodiazole,2,3,3-trimethylindolinine, 2,3,3-trimethyl-4,5-benzo-3H-indolinine, 3-and 4-picoline, lipidine, chinadine and 9-methylacridine derivativeswith the general formula IIa or IIb or IIc

and wherein X is selected from the group consisting of O, S, Si, N-alkyland C(alkyl)₂, n is 0, 1, 2 or 3, R¹ to R¹⁴ are independently selectedfrom the group consisting of hydrogen, alkyl, alkoxy, cycloalkyl, linearor branched alkenyl, cycloalkenyl, aryl, heteroaryl, heterocycle,hydroxy, carboxy, amine, alkyl-substituted amine and cyclic amine and/ortwo or more fragments in ortho-position to each other, for example R¹⁰and R¹¹ or R⁴, R⁵ and R⁶, together form another cycloalkyl ring or ringsystem, heterocyclic ring or ring system, heteroaryl ring system oraromatic ring or ring system.
 39. The use of claim 38, wherein themarker dye is defined as in any of claims 10 to
 21. 40. Use of apolymethine of the general formula (I) for the analysis of HDL and/orLDL subclass patterns in a sample by labelling lipoproteins with thepolymethine of the general formula I

wherein Z is a substituted derivative of benzooxazole, benzodiazole,2,3,3-trimethylindolinine, 2,3,3-trimethyl-4,5-benzo-3H-indolinine, 3-and 4-picoline, lipidine, chinadine and 9-methylacridine derivativeswith the general formula IIa or IIb or IIc

and wherein X is selected from the group consisting of O, S, Si, N-alkyland C(alkyl)₂, n is 0, 1, 2 or 3, R¹ to R¹⁴ are independently selectedfrom the group consisting of hydrogen, alkyl, alkoxy, cycloalkyl, linearor branched alkenyl, cycloalkenyl, aryl, heteroaryl, heterocycle,hydroxy, carboxy, amine, alkyl-substituted amine and cyclic amine and/ortwo or more fragments in ortho-position to each other, for example R¹⁰and R¹¹ or R⁴, R⁵ and R⁶, together form another cycloalkyl ring or ringsystem, heterocyclic ring or ring system, heteroaryl ring system oraromatic ring or ring system.
 41. The use of claim 40, wherein themarker dye is defined as in any of claims 10 to 21.